Effect of salicylate on expression of flagella by Escherichia coli and Proteus, Providencia, and Pseudomonas spp.

Osmotic stress, salicylate, and Mar (multiple antibiotic resistance) mutation are known to block the expression of the OmpF porin. Since these conditions have also been shown to inhibit the expression of P and CFA fimbriae in Escherichia coli, we speculated that they might affect the expression of flagella as well. Hyperosmotic conditions have been shown to block the synthesis of flagellin and expression of flagella in E. coli (C. Li, C. J. Louise, W. Shi, and J. Adler, J. Bacteriol. 175:2229-2235, 1993). In the current study, sodium salicylate was found to inhibit the motility of E. coli, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, and Providencia stuartii in a reversible, concentration-dependent manner. Swarming did not occur at 20 mM sodium salicylate. Salicylate also blocked the synthesis of flagellin in E. coli. Phenotypic Mar mutants of E. coli derived from motile strains were amotile. Flagella were markedly reduced as determined by scanning electron microscopy when P. mirabilis was grown in broth containing 20 mM salicylate. Salicylate had no apparent effect, however, on expression of a 40-kDa porin protein in P. mirabilis. This finding suggests that the noted effect of salicylate on Proteus spp. may be mediated through a mechanism other than porin production or that the Proteus porin may not be analogous to OmpF in E. coli. Salicylate decreased the motility of Pseudomonas cepacia but had no effect on Pseudomonas aeruginosa. The exact mechanism by which salicylate exerts its effect is not known, but it appears to be related to osmoregulation.     

The 987P fimbrial gene cluster of enterotoxigenic Escherichia coli is plasmid encoded.

A clone containing the 987P fimbrial gene cluster was selected from a cosmid library of total DNA of the prototype Escherichia coli strain 987 by using 987P-specific antiserum. A subclone of 12 kilobases containing all of the genes required for fimbrial expression on a nonfimbriated K-12 strain of E. coli and a DNA fragment internal to the fimbrial subunit gene were used to probe the prototype strain and various isolates of 987P-fimbriated enterotoxigenic E. coli. All strains had several plasmids, as shown by agarose gel electrophoresis, and each of five strains which expressed 987P fimbriae showed a plasmid of 35 to 40 megadaltons (MDa) hybridizing to both 987P-specific probes. Hybridization to restricted DNA of strain 987 supported a plasmid origin for the cloned 987P gene cluster. Moreover, an isogenic strain which had lost its 35-MDa plasmid was no longer capable of synthesizing fimbrial subunits, but regained fimbrial expression after reintroduction of the TnphoA (Tn5 IS50L::phoA)-tagged 35-MDa plasmid. Absence of fimbrial subunit synthesis in K-12 strains transformed with the 35-MDa plasmid alone suggested the requirement of regulatory elements existing in strain 987 but missing in K-12 strains. A probe for the heat-stable enterotoxin STIa hybridized in each of the 987P-fimbriated strains to the plasmid containing the 987P genes and in most of these strains to an additional plasmid which contained the gene for the heat-stable enterotoxin STII. Occurrence of the 987P and STIa genes on the same replicon correlates with epidemiological observations, STIa being the most prevalent toxin produced by 987P-fimbriated E. coli.     

Ultrastructural study of adhesion of enterotoxigenic Escherichia coli to erythrocytes and human intestinal epithelial cells

The adhesion to erythrocytes and human intestinal epithelial cells of enterotoxigenic Escherichia coli strains H10407, B2C, and H10407P, expressing colonization factor antigen I (CFA/I), CFA/II, and type 1 fimbriae, respectively, was examined by electron microscopy. CFA and type 1 fimbriae were visualized by negative staining in thin sections after en bloc staining with ruthenium red and by immune labeling with antisera raised against purified fimbriae. By negative and ruthenium red staining, CFA/I, CFA/II, and type 1 fimbriae were indistinguishable and appeared as approximately 7-nm-diameter hollow cylindrical structures up to 1.5 micron in length; strain B2C also produced 2- to 3-nm-diameter flexible fibrillar fimbriae. Bacteria producing CFA/I, CFA/II, and type 1 fimbriae adhered to and agglutinated human, bovine, and guinea pig erythrocytes, respectively; CFA/I and CFA/II also mediated attachment of bacteria to the brush border of isolated human duodenal enterocytes. Electron microscopy of agglutinated erythrocytes and enterocytes with adherent bacteria showed, in each case, that bacterial adhesion involved the formation of many interactions between the tips of fimbriae and receptors on the erythrocyte or enterocyte brush border membrane. Immune labeling allowed different fimbrial antigens mediating bacterial attachment to human enterocytes to be identified.     

Avian P1 antigens inhibit agglutination mediated by P fimbriae of uropathogenic Escherichia coli.

Whole egg white from pigeon, dove, and cockatiel eggs, as well as the ovomucoid fraction of pigeon egg white, exhibited strong P1 antigenic activities and inhibited agglutination of human P1 erythrocytes and of digalactoside-coated latex beads by P-fimbriated Escherichia coli strains. In contrast, chicken egg white exhibited only weak P1 antigenic activity and had little impact on P-fimbrial agglutination. These preparations did not affect hemagglutination by E. coli strains expressing mannose-resistant adhesins other than P fimbriae, i.e., Dr, F1845, and S adhesins. Human anti-P1 serum diminished the P-fimbrial inhibitory activities of pigeon egg white and pigeon ovomucoid. Pigeon ovomucoid was equipotent on a molar basis with globoside, and the pigeon, dove, and cockatiel egg white preparations were equipotent with each other in P-fimbrial inhibition. Incubation of p erythrocytes in whole egg whites or in pigeon ovomucoid did not render them agglutinable by P-fimbriated bacteria, whereas incubation in globoside did. These data demonstrate that whole egg whites (and their ovomucoid fraction) from members of the families Columbidae (pigeons and doves) and Psittacidae (parrots) specifically and potently inhibit P-fimbrial agglutination, probably by providing P1 antigen as a receptor for the P-fimbrial adhesin. Avian egg white preparations may facilitate adhesin characterization of wild-type uropathogenic strains and may useful in preventing upper urinary tract infections due to P-fimbriated E. coli.     

New fimbrial antigenic type (E8775) that may represent a colonization factor in enterotoxigenic Escherichia coli in humans.

An enterotoxigenic strain of Escherichia coli O25:H42 (strain E8775), isolated from a patient in Bangladesh with diarrhea, caused mannose-resistant hemagglutination (MRHA) of human and bovine erythrocytes. The strain did not show slide agglutination or immunodiffusion precipitin lines with antiserum specific for the colonization factor antigen CFA/I or CFA/II. A variant E. coli strain, E8775-B, did not cause MRHA or produce enterotoxin. Electron microscopy revealed the presence of fimbriae on the surface of strain E8775 but not strain E8775-B. When strain E8775 was grown at 22 degrees C, it became MRHA negative and fimbriae were absent. An antiserum prepared against strain E8775 was absorbed with strain E8775-B to make an antiserum specific for the fimbrial antigen. Using this absorbed antiserum, we found the fimbrial antigen in 48 of 742 enterotoxigenic E. coli strains. The 48 strains belonged to serogroups O25, O115, and O167. It is suggested by analogy to the properties of previously described colonization factors that these fimbriae may play a part in the colonization of the intestinal epithelium.     

Roles of the pap- and prs-encoded adhesins in Escherichia coli adherence to human uroepithelial cells.

In this study, we reexamined the structural prerequisites for the attachment of P-fimbriated Escherichia coli to human urinary tract epithelial cells. The epithelial cells were obtained from A1P1 nonsecretor individuals, who express the globoseries of glycolipids without the ABH blood group determinants, and from A1P1 secretor individuals, who in addition express globo-A, a receptor for the prsJ96 adhesin. The wild-type E. coli strains J96, AD110, and IA2 and the recombinant clones HB101 papJ96, HB101 prsJ96, HB101 papIA2, and HB101 papAD110 were tested for binding. They expressed P fimbriae, as defined by P blood group-dependent agglutination of human erythrocytes of the globoseries, but differed in reactivity with galactose alpha 1-4galactose beta (Gal alpha 1-4Gal beta)-latex beads, isolated glycolipids of the globoseries, sheep erythrocytes, and uroepithelial cells. Three different patterns of binding were represented among the recombinant clones. HB101 papIA2 and HB101 papAD110 agglutinated sheep erythrocytes and Gal alpha 1-4Gal beta-latex beads and attached to both secretor and nonsecretor epithelial cells. HB101 prsJ96 agglutinated sheep erythrocytes, reacted poorly with Gal alpha 1-4Gal beta-latex beads, and attached to A1 secretor but not to A1 nonsecretor epithelial cells. HB101 papJ96 agglutinated Gal alpha 1-4Gal beta-latex beads but not sheep erythrocytes and attached poorly to human uroepithelial cells. The receptors relevant for adhesion were analyzed by inhibition with glycolipids in suspension. The sheep erythrocyte agglutination and attachment to secretor and nonsecretor epithelial cells of HB101 papIA2 and HB101 papAD110 were inhibited by globotetraosylceramide, while the Forssman glycolipid had no effect. The sheep erythrocyte reactivity and attachment to secretor epithelial cells of HB101 prsJ96 were inhibited by the Forssman glycolipid. These results permitted three conclusions. First, the expression of functionally active Gal alpha 1-4Gal beta-specific adhesins, as in HB101 papJ96, was not sufficient to make E. coli competent to attach to human uroepithelial cells. Attachment required P fimbriae of the papIA2 or papAD110 type. Second, the sheep erythrocyte reactivity of P-fimbriated strains could not be attributed solely to recognition of the Forssman glycolipid and may not be used to define the prsJ96-encoded phenotype. Third, the P-fimbrial adhesins which mediate secretor state-independent attachment to human uroepithelial cells recognized receptor epitopes provided by globotetraosylceramide.     

Analysis of P fimbriae on Escherichia coli O2, O4, and O6 strains by immunoprecipitation.

P fimbriae on Escherichia coli O2, O4, and O6 strains were analyzed by immunoprecipitation. Fimbrial extracts were prepared from a total of 35 strains and tested for precipitation with four anti-P-fimbria sera. The overall fimbrial composition of the strains was related to the O:K:H serotype, and two to three P fimbrial variants per strain were found on most of the O4 and some of the O6 strains. The O2 strains, in contrast, showed only one antigenic variant of P fimbriae per strain, which was serologically unrelated to those of the O4 and O6 strains. The results stress the multiplicity and serological complexity of E. coli P fimbriae.     

A plasmid of enterohemorrhagic Escherichia coli O157:H7 is required for expression of a new fimbrial antigen and for adhesion to epithelial cells.

Of 14 strains of Escherichia coli O157:H7 isolated from patients with hemorrhagic colitis or hemolytic uremic syndrome that were examined for fimbriae, the presence of plasmids, and the ability to adhere to intestinal cells, 13 possessed a 60-megadalton plasmid and were fimbriated as assessed by electron microscopy. These strains adhered to Henle 407 intestinal cells but not to HEp-2 cells or erythrocytes. Three strains were cured of the plasmid and thereafter failed to express fimbriae and lost the ability to adhere to intestinal cells. Conversely, E. coli K-12 transformed with the 60-megadalton plasmid from each of the three strains produced fimbriae and was able to adhere to intestinal cells. A single fimbrial subunit of 16 kilodaltons was observed when purified fimbriae from the transformants and from the 60-megadalton plasmid-containing E. coli O157:H7 strains were disaggregated and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antisera raised against one preparation of the purified fimbriae reacted strongly with 12 of 14 O157:H7 isolates in an agglutination assay and with purified fimbrial preparations from five E. coli O157:H7 strains in an enzyme-linked immunosorbent assay.     

Characterization of genes encoding type 1 fimbriae of Klebsiella pneumoniae, Salmonella typhimurium, and Serratia marcescens.

With a minicell system, the organization of genes encoding type 1 fimbriae of Salmonella typhimurium, Klebsiella pneumoniae, and Serratia marcescens was determined. In all cases multiple gene products were necessary for the phenotypic expression of fimbriae; thus fimbrial expression in these strains is similar to that in Escherichia coli. The type 1 fimbrial subunit gene was detected by the ability of its product to react with specific antiserum. At least six genes were found to be involved in the expression of type 1 fimbriae by S. typhimurium, and at least four genes constituted the fimbrial gene cluster of K. pneumoniae. In the case of S. marcescens, a minimum of three detectable polypeptides was required for the production of fimbriae. Also, a gene probe consisting in part of nucleotide sequences from the E. coli fimbrial subunit gene hybridized to a discrete DNA fragment derived from the plasmid encoding K. pneumoniae fimbriae. Such a fragment was assumed to contain a gene encoding the structural component of the type 1 fimbriae. Each of the three cloned systems encoded a number of polypeptides which varied in size; thus, the organization and molecular weight of fimbrial accessory proteins of each genus were not identical.     

Characterization of P fimbriae on O1, O7, O75, rough, and nontypable strains of Escherichia coli.

P fimbriae of 37 uropathogenic Escherichia coli O1:K1, O7:K1, O22, O75, rough:K1, and nontypable strains were characterized by immunoprecipitation with 14 fimbria-specific rabbit antisera. The fimbrial composition of these strains, as reflected by the apparent molecular weights of the fimbrial peptides, was correlated with the O serogroup of the strains, but serological cross-reactivity of P fimbriae of different E. coli serogroups was frequently observed. The genetic clonal relationships of the strains were analyzed by determining the electrophoretic types, based on 18 chromosomally encoded enzymes. Among the O1:K1 strains, the same P-fimbrial variants occurred on strains that were either closely related or very distinct in their electrophoretic types, indicating that the P fimbriae have evolved in association with the O and K antigens. In contrast, certain O7:K1 and R:K1 strains as well as some O22 and O75 strains were genotypically identical and shared similar P-fimbrial variants, which differed serologically from those of other E. coli serogroups. Our results show that, despite the structural variability seen in electrophoretic analysis of P fimbriae of different serogroups, many P-fimbrial variants share common antigenic determinants that are recognized by rabbit antisera. Based on immunoprecipitation analyses, three anti-P-fimbria sera have now been identified that react with P fimbriae of 82 of 84 uropathogenic E. coli strains characterized in Finland.     

Identification of a new fimbrial structure in enterotoxigenic Escherichia coli (ETEC) serotype O148:H28 which adheres to human intestinal mucosa: a potentially new human ETEC colonization factor.

Three important fimbrial colonization factor antigens (CFAs) designated CFA/I, CFA/II, and E8775 were identified originally in some human enterotoxigenic Escherichia coli (ETEC) strains because of their mannose-resistant hemagglutination properties. To identify CFA, in strains lacking mannose-resistant hemagglutination properties we exploited the ability of human ETEC strains to adhere to human proximal small intestinal mucosa. ETEC strain B7A (O148:H28) was selected for study because it belongs to an epidemiologically important serotype and does not produce a known CFA, and yet it is known to be pathogenic and cause diarrheal disease in human volunteers. Results of an human enterocyte adhesion assay indicated that some bacteria in cultures of B7A produced adhesive factors. To select for such bacteria, cultured human duodenal mucosal biopsy samples were infected with B7A for up to 12 h, after which time a large percentage of the mucosal surface became colonized by bacteria. A new fimbrial structure morphologically distinct from CFA/I, CFA/II, and E8775 fimbriae and consisting of curly fibrils (approximately 3 nm in diameter) was readily identified when bacteria were subcultured from the mucosa and examined by electron microscopy. Identical fimbriae were produced by ETEC strain 1782-77 of the same serotype. Identification of these fimbriae only on bacteria subcultured from human intestinal mucosa strongly suggests that they promote mucosal adhesion of ETEC serotype O148:H28 and thus represent a potentially new human ETEC CFA.     

Characterization of 20K fimbria, a new adhesin of septicemic and diarrhea-associated Escherichia coli strains, that belongs to a family of adhesins with N-acetyl-D-glucosamine recognition.

Bovine septicemic Escherichia coli 31A agglutinates bovine, rabbit, and human erythrocytes and adheres in vitro to the brush border of bovine or ovine intestinal epithelial cells and to the human colon carcinoma Caco-2 cell line. The adhesion and hemagglutination of E. coli 31A are mediated by a chromosome-encoded fimbrial adhesin serologically distinct from known fimbrial adhesins found in enterotoxigenic and septicemic bovine E. coli strains. By electron microscopy studies the fimbriae designated 20K were observed as fine flexible filaments (diameter, 3 nm) and the purified major fimbrial subunit appeared with an apparent molecular mass of 20,000 Da. Western blot (immunoblot) analysis, N-terminal sequence alignment, and amino acid composition revealed a high homology with the N-acetyl-D-glcosamine-specific G fimbria of human uropathogenic E. coli and with fimbriae belonging to the F17 family produced by bovine enterotoxigenic and invasive E. coli strains. Immunological study revealed that 20K fimbria was closely related to G fimbria and represents a serological variant of F17 fimbria. Hemagglutination and adhesion inhibition assays demonstrated that 20K, G, and F17 fimbriae bind to an N-acetyl-D-glucosamine-containing receptor, but each probably binds to different oligosaccharide sequences or different receptors on host tissues. 20K fimbriae were produced by a limited group of clonally related strains with the unusual m-inositol-positive phenotype and appeared highly associated with the plasmid-mediated virulence factor. An examination of natural occurrence of 20K fimbriae among a large collection of human and animal pathogenic E. coli showed that 20K fimbria is the prominent adhesin among bovine septicemic E. coli isolated from European countries.     

Hemagglutinating and chemotactic properties of synthetic peptide segments of fimbrial protein from Porphyromonas gingivalis.

Porphyromonas gingivalis 381 fimbriae, their synthetic peptide segments, and lipopolysaccharide (LPS) were examined for hemagglutinating and migration-stimulating activities. P. gingivalis 381 fimbriae clearly caused hemagglutination, and several oligopeptide segments such as FP381(61-80), FP381(171-185), and FP381(302-321), corresponding to the amino acid residue numbers based on the amino acid sequence of fimbrillin proposed by Dickinson et al. (D. P. Dickinson, M. A. Kubiniec, F. Yoshimura, and R. J. Genco, J. Bacteriol. 170:1658-1665, 1988), were also demonstrated to agglutinate erythrocytes although less effectively than the native fimbriae. Furthermore, P. gingivalis 381 LPS but not Escherichia coli O55:B5 LPS definitely exhibited hemagglutination. P. gingivalis fimbriae as well as their synthetic peptides possessing hemagglutinating activity enhanced the chemotaxically induced migration of human peripheral blood monocytes. The results of the analyses using synthetic peptide FP381(61-80), its related compounds, and an analog suggested that the amino acid sequence XLTXXLTXXNXX within fimbrial protein molecules may play an important role structurally in the attachment of the protein to host cells such as erythrocytes and monocytes.     

Fimbriation, capsulation, and iron-scavenging systems of Klebsiella strains associated with human urinary tract infection.

Thirty-two strains of Klebsiella pneumoniae and seven strains of Klebsiella oxytoca isolated from urinary tract infections in elderly adults were analyzed for capsular antigens, iron-scavenging systems, and fimbriation. All strains were capsulated. Twenty-seven different K antigens were identified among the strains, with no particular antigen dominating. All strains produced the iron-scavenging system enterochelin as analyzed by bioassay and DNA hybridization. In contrast, the aerobactin iron-sequestering system was not detected in any of the strains. All strains caused hemagglutination of tannin-treated human erythrocytes and reacted with an anti-type 3 fimbriae antiserum as well as in DNA hybridization with a type 3 fimbria-specific probe, indicating that the Klebsiella strains possessed this fimbrial type. Possession of type 1 fimbriae was analyzed by agglutination tests and by hybridization with DNA probes from two distinct Klebsiella type 1 fimbria gene clusters. Phenotypic expression of the type 1 fimbriae was found in 29 of 32 K. pneumoniae strains, whereas 30 strains reacted with either of the two type 1 fimbrial cluster DNA probes. In K. oxytoca, however, only three of seven strains expressed type 1 fimbriae and reacted with the DNA probes. The type 3 fimbriae were found to bind to a fraction of epithelial cells exfoliated in normal human urine, whereas the type 1 fimbriae bound strongly to urinary slime. No inhibitors of type 3 fimbrial binding were detected in human urine.     

Purification and characterization of the CFA/I antigen of enterotoxigenic Escherichia coli.

The fimbral colonization factor antigen CFA/I of enterotoxigenic Escherichia coli was purified and characterized. The initial purification step was release of these fimbriae from the bacterial cells by homogenization with a Waring blender. Common fimbriae and flagellar antigen were avoided by careful control of growth conditions and the use of a nonmotile (H-) mutant of the prototype strain H-10407 (O78:H11). The essential purification steps were membrane filtration (Millipore Corp.), ammonium sulfate fractionation, and negative diethylaminoethyl-Sephadex column chromatography. Yields were approximately 4.0 mg of CFA/I protein per g (wet weight) of bacteria. Purified CFA/I is a fimbrial molecule 7.0 nm in diameter and has an average molecular weight of 1.6 X 10(6), as determined by sedimentation equilibrium. CFA/I is a polymer of identical subunits of molecular weight 23,800 with an N-terminal valine, 37% hydrophobic amino acid residues, and 11 residues of proline per mol. The purified antigen retains its morphology, antigenicity, and biological activity. Purified antigen retains its morphology, antigenicity, and biological activity. Purified CFA/I exhibits mannose-resistant hemagglutination of human group A, bovine, and chicken erythrocytes, as do CFA/I-positive bacteria. This was demonstrated by sensitizing latex microbeads with the purified antigen since cell-free CFA/I fimbriae do not hemagglutinate erythrocytes. Thus, CFA/I detached from the bacteria are monovalent; however, purified CFA/I antigen retains an affinity for the epithelial cells of rabbit small intestine and blocks adhesion of CFA/I-positive bacteria. These results demonstrate that purified CFA/I is a good candidate for use in an oral vaccine for immunoprotection against diarrhea caused by CFA/I-positive enterotoxigenic E. coli.     

Purification and characterization of serotype 6 fimbriae from Bordetella pertussis and comparison of their properties with serotype 2 fimbriae.

Fimbriae were removed from Bordetella pertussis (serotype 1.3.6) by mechanical shearing and purified by precipitation with ammonium sulfate, pH-dependent precipitation at pH 7.4, followed by two successive extractions of the precipitated fimbriae with 4 M urea. By electron microscopy, the precipitated fimbriae appeared as aggregated bundles of long, relatively straight filaments which were disaggregated to individual flexuous filaments at pH 10.5. These purified fimbriae were identified as serotype 6 agglutinogens, since antibody to the purified fimbriae agglutinated B. pertussis strains serotyped as 1.3.6, 1.2.3.6, or 1.2.3.4.6 but did not agglutinate strains of serotype 1.2.3.4, 1.2.3, or 1.3. In contrast, antibody to serotype 2 fimbriae only agglutinated B. pertussis strains containing serotype 2 agglutinogen. Purified type 6 and 2 fimbriae were found to be weakly cross-reactive by enzyme-linked immunosorbent assay, using polyclonal antibody to each type of fimbria. In an immunoblot assay, polyclonal antibodies to a 22,000-dalton subunit of fimbriae from B. bronchiseptica reacted strongly with the type 2 fimbrial subunit of B. pertussis, but only weakly with the type 6 subunit. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein subunit of the type 6 fimbriae migrated with a molecular weight of 21,500, whereas the type 2 fimbrial subunit had a molecular weight of 22,000. The two types of subunits had similar amino acid compositions and showed amino-terminal sequence homology in 15 of 21 amino acids. The amino-terminal amino acid sequences of the B. pertussis fimbriae were distinct from those reported for fimbriae from other gram-negative bacteria. Neither the type 6 nor the type 2 fimbriae caused hemagglutination when assayed with several types of erythrocytes.     

Cloning, expression, and characterization of fimbrial operon F9 from enterohemorrhagic Escherichia coli O157:H7

Recent transposon mutagenesis studies with two enterohemorrhagic Escherichia coli (EHEC) strains, a sero- type O26:H- strain and a serotype O157:H7 strain, led to identification of a putative fimbrial operon that promotes colonization of young calves (1 to 2 weeks old). The distribution of the gene encoding the major fimbrial subunit present in O-island 61 of EHEC O157:H7 in a characterized set of 78 diarrheagenic E. coli strains was determined, and this gene was found in 87.2% of the strains and is therefore not an EHEC-specific region. The cluster was amplified by long-range PCR and cloned into the inducible expression vector pBAD18. Induced expression in E. coli K-12 led to production of fimbriae, as demonstrated by transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The fimbriae were purified, and sera to the purified major subunit were raised and used to demonstrate expression from wild-type E. coli O157:H7 strains. Induced expression of the fimbriae, designated F9 fimbriae, was used to characterize binding to bovine epithelial cells, bovine gastrointestinal tissue explants, and extracellular matrix components. The fimbriae promoted increases in the levels of E. coli K-12 binding only to bovine epithelial cells. In contrast, induced expression of F9 fimbriae in E. coli O157:H7 significantly reduced adherence of the bacteria to bovine gastrointestinal explant tissue. This may have been due to physical hindrance of type III secretion-dependent attachment. The main F9 subunit gene was deleted in E. coli O157:H7, and the resulting mutant was compared with the wild-type strain for colonization in weaned cattle. While the shedding levels of the mutant were reduced, the animals were still colonized at the terminal rectum, indicating that the adhesin is not responsible for the rectal tropism observed but may contribute to colonization at other sites, as demonstrated previously with very young animals.     

Adhesive fimbriae associated with porcine enterotoxigenic Escherichia coli of the O141 serotype.

Enterotoxigenic Escherichia coli of the O141 serotype, isolated from piglets with postweaning coliform enteritis but producing none of the characterized adhesive fimbriae, was examined for fimbrial production by transmission electron microscopy. Two strains that produced numerous fimbriae were chosen for further characterization. The fimbriae were isolated and purified and had a subunit molecular weight of 17,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using antiserum raised against this protein, we have shown it to be specific for the 17,000-molecular-weight band by immunoblotting and to be directed against the fimbriae by immunoelectron microscopy. These fimbriae were not produced when the bacteria were grown at 18 degrees C and did not show any mannose-resistant hemagglutination of the erythrocytes tested. We propose that these are a new type of adhesive fimbriae associated with porcine enterotoxigenic E. coli of the O141 serotype.     

Role of Escherichia coli P fimbriae in intestinal colonization in gnotobiotic rats.

Adherence via P fimbriae is associated with long-term persistence of Escherichia coli in the human large intestine, but a causal relationship has not been proven. In the present study, germfree rats were colonized with a mixture of two isogenic E. coli strains, one P fimbriated and the other type 1 fimbriated. Both types of fimbriae conferred adherence to rat colonic epithelial cells. With two mutant strains from a pyelonephritogenic isolate of serotype O75:K5:H-, the P-fimbriated strain 824 attained much higher numbers than its type 1-fimbriated counterpart when colonized in vivo for 2 weeks (10(10) versus 10(6) bacteria per g, respectively; P < 0.0001). The expression of P fimbriae by 824 was also retained during colonization. With transformant isogenic strains obtained from a normal fecal isolate incapable of phase variation, no benefit of P fimbriae was seen and most bacteria lost their plasmids during in vivo colonization. When the pyelonephritogenic mutant and fecal transformant strains were combined, the former colonized at high levels while the latter were suppressed. In contrast, no suppression was seen when the transformant E. coli strains colonized in combination with Lactobacillus acidophilus or Peptostreptococcus sp. The results indicate that P fimbriae, but also other bacterial traits linked to uropathogeneicity, could play an important role for persistence in the gut normal microbiota. Neither P nor type 1 fimbriae seemed to contribute to the ability to translocate to the mesenteric lymph nodes.     

Three-dimensional structure of fimbriae determines specificity of immune response.

We recently described how a fraction of isolated fimbriae from a multifimbriated strain of Escherichia coli O7:K1:H6 (WF96) could be subdivided by sequential disaggregation in disrupting agents into individual subunits with different molecular weights. In this study, antibodies were raised in rabbits against these isolated fimbrial subunits and against purified intact WF96 fimbriae. These sera were tested by Western blot analysis or by enzyme-linked immunosorbent assays for reactivity against the following antigens: intact WF96 fimbriae, dissociated WF96 fimbriae, dissociated and reaggregated WF96 fimbriae, the WF96 21K fimbrial subunit, reaggregated WF96 21K subunits, the WF96 16K subunits, reaggregated WF96 16K subunits, intact fimbriae from four other E. coli strains, and deaggregated fimbriae from these strains. We found that antibody against intact WF96 fimbriae only reacted strongly with intact WF96 fimbriae, depolymerized and reaggregated WF96 fimbriae, or reaggregated fimbrial subunits; no reactions were evident with intact fimbriae from four other E. coli strains. Conversely, antisera prepared against the WF96 16K subunit and against the WF96 21K subunit did not react with intact WF96 fimbriae or with depolymerized and reaggregated WF96 fimbriae, but did react with homologous isolated subunits. One cross-reaction between fimbrial subunits was apparent: anti-WF96 16K subunit bound to a 21K subunit of deaggregated fimbriae, from another E. coli strain. Taken together, the findings indicate that the three-dimensional structure of the fimbrial preparation used to immunize animals determines the specificity of the immune response.